SEVENTEENTHANNUALRESEARCHSYMPOSIUM

ANIMALMOLECULARANDCELLULARBIOLOGYGRADUATEPROGRAM

UNIVERSITYOFFLORIDA

InternationalPalmsResort&ConferenceCenterCocoaBeach,Florida

April12‐13,2019

WELCOMEOurlocationthisyearisnotablenotonlybecausewegettoenjoythebeautyoftheAtlanticOceanbutbecausewearemeetingat thehomeof theKennedySpaceCenter,oneof themostconsequentiallocationsforscienceintheUnitedStates.Itwasfromherethathumansfirst went to the Moon. It is often overlooked but the National Aeronautics and SpaceAdministration(NASA)isalsoinvolvedinbiologicalresearch.TheUniversityofFloridaisapartnerinthatresearchandmaintainsalaboratorybuildingattheKennedySpaceCentercalledtheSpaceLifeSciencesLab.ThephotoonthecoverisofSpaceShuttleEndeavouronmissionSTS‐126asitpreparedtodockwiththeInternationalSpaceStationonNovember16,2007.OnboardwerehundredsofbovineembryossentupbyresearchersfromtheUFDept.ofAnimalSciences.Thisyear,forthefirsttime,thepresentationshavenotbeenorganizedbytopicbutinsteadtomaintainintellectualdiversityineachsession.Inrecognitionofourlocation,thesessionsarenamedafterimportantNASAspaceprograms.WearepleasedtohaveDr.BrianO’NeillastheAMCBDistinguishedLecturer.Wearealsograteful for those who support the AMCB during the year. We receive significant andsustained funding from the College of Agricultural and Life Sciences and the L.E. “Red”Larson Endowment. The symposium itself also received support from the University ofFloridaOfficeofResearchand,forthefirsttime,BalchemCorporation.Duringtheyear, thedailyoperationsof theAMCBaremaintainedbyReneeParks‐James.ThankyouRenee. Inaddition, thesymposiumwouldnothavebeenpossiblewithout thetirelesseffortsofLizJannamanoftheDept.ofAnimalSciences.AlmosteveryaspectofthelocalarrangementswasorganizedbyLiz.Pleasethankherwhenyouseeher.Asalways,theAMCBisallaboutconnections–anditishopedthatdiscussionsduringthebreaksandotherinformaloccasionsduringthesymposiumwillleadtosomenewinsightsandnewcollaborations.OnbehalfofallthefacultyoftheAMCB,welcometothe17thAnnualResearchSymposiumoftheAMCB.Havesomefun,buildsomeconnections,andlearn!PeteHansen,DirectorJohnDriver,Co‐Director

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ACKNOWLEDGMENTS

ThefacultyandstudentsoftheAMCBProgramthankthefollowingforsupportofthe

17thAnnualResearchSymposium

Dr.R.ElaineTurner,Dean,CollegeofAgricultural&LifeSciences,UniversityofFlorida

Dr.DavidNorton,VicePresidentforResearch,UniversityofFlorida

L.E.“Red”LarsonEndowment

BalchemCorporation

AppreciationisalsoexpressedtothosewhohavesupportedtheAMCBProgramthroughouttheyear

Dr.RalucaMateescu,GraduateCoordinator,AnimalMolecularandCellularBiology

GraduateProgram,UniversityofFlorida

Ms.ReneeParks‐James,ProgramAssistant,Dept.ofAnimalSciences,UniversityofFlorida

Dr.GeoffreyE.Dahl,ProfessorandChair,Dept.ofAnimalSciences,UniversityofFlorida

ElizabethJannaman,Dept.ofAnimalSciences

PeterHansenandJohnDriver,DirectorandCo‐DirectoroftheAMCBGraduateProgram

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2019AMCBDISTINGUISHEDLECTURER

BrianT.O’Neill,MD,PhDUniversityofIowaCarverCollegeofMedicine

Brian T. O’Neill is an assistant professor and endocrinologist at the University of Iowa,CarverCollegeofMedicine.HereceivedhisMD/PhDfromtheUniversityofUtah,wherehecompletedhisthesisworkwithDr.E.DaleAbelontheregulationofcardiacmetabolismbyPI3‐Kinase and Akt. Dr. O’Neill then did endocrinology fellowship and post‐graduatetraining at Beth Israel DeaconessMedical Center and Joslin Diabetes Center, affiliates ofHarvardMedicalSchool.UnderthementorshipofDr.C.RonaldKahn,Dr.O’NeilldiscoveredthatinsulinreceptorsandIGF‐1receptorscoordinatemusclegrowthandsuppressatrophybyregulatingFoxOtranscriptionfactors.Muscle atrophy and decreased muscle strength are under‐appreciated complications ofuncontrolleddiabetesinolderpopulations,whichcanleadtodisabilityorevendeathfromsurgery or severe illness.Dr. O’Neill’s lab usesmouse and cellmodels to investigate theroles of insulin signaling and FoxO regulation in the control of muscle strength andmitochondrialmetabolism, in hopes of identifying the intracellular signalingdefects thatoccur in the diabetic state. The ultimate goal of Dr. O’Neill’s research is to providetherapeutictargetsthatbenefitmuscleenergyproductionindiabetes,speedrecoveryaftersevereillnessorsurgery,andpreventdisability.

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AMCBFACULTYMarioBinelli.DepartmentofAnimalSciences

JohnJ.Bromfield,DepartmentofAnimalSciences

SamanthaA.Brooks,DepartmentofAnimalSciences

MaryB.Brown,DepartmentofInfectiousDiseasesandPathology

GeoffreyE.Dahl,DepartmentofAnimalSciences

JohnDriver,DepartmentofAnimalSciences

TimothyJ.Hackmann,DepartmentofAnimalSciences

PeterJ.Hansen,DepartmentofAnimalSciences

KwangCheolJeong,DepartmentofAnimalSciences

Qiu‐XingJiang,DepartmentofMicrobiology&CellScience

MaureenKeller‐Wood,DepartmentofPharmacodynamics

JimenaLaporta,DepartmentofAnimalSciences

RalucaMateescu,DepartmentofAnimalSciences

CorwinNelson,DepartmentofAnimalSciences

JoséE.P.Santos,DepartmentofAnimalSciences

CharlesE.Wood,DepartmentofPhysiologyandFunctionalGenomics

EmeritusFacultyLokengaBadinga,DepartmentofAnimalSciences

WilliamC.Buhi,DepartmentsofObstetrics&Gynecology,AnimalSciences

KennethC.Drury,DepartmentofObstetrics&Gynecology

MichaelJ.Fields,DepartmentofAnimalSciences

DanielC.Sharp,DepartmentofAnimalSciences

WilliamW.Thatcher,DepartmentofAnimalSciences

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CURRENTAMCBSTUDENTSPhDStudentsThiagoF.Amaral(Advisor:Hansen)

UsmanArshad(Advisor:Santos)

MackenzieJDickson(Advisor:Bromfield)

EliabEstradaCortes(Advisor:Hansen)

ZairaEstradaReyes(Advisor:Mateescu)

WeihongGu(Advisor:Driver)

AliHusnain(Advisor:Santos)

MelanyDDarlingMadrid(Advisor:Driver)

PaulaCardosoMolinari(Advisor:Bromfield)

MichaelB.Poindexter(Advisor:NelsonandSantos)

FroylanSosaHernandez(Advisor:Hansen)

AchillesVieiraNeto(Advisor:Santos)

ChengYe(Advisor:Driver)

BoZhang(Advisor:Hackmann)

RoneyZimpel(Advisor:Santos)

MSStudents

SamanthaBohm(Advisor:Nelson)

SenaField(Advisor:Laporta)

CourtneyMcCourt(Advisor:Hackmann)

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GRADUATESOFTHEAMCB,2018‐2019

BethanyDadoSenn,MS(AdvisorJLaporta)CurrentlyanAnimalSciencesdoctoralstudentintheLaportalaboratory,UniversityofFloridaLailaA.Ibrahim,MS(Advisor:JJBromfield)CurrentlyafacultymemberofUniversityofBenghazi;willreturntoUFinthefallof2019JasonA.Rizo,MS(Advisor:JJBromfield)Currentlyadoctoralstudent,UniversityofMissouriMercedesKweh,PhD(advisor:C,Nelson)CurrentlyapostdoctoralfellowatUniversityofMiamiCancerEpigeneticTrainingProgramGuanYang,PhD(AdvisorJDriver)CurrentlyapostdoctoralfellowintheDeptofPathology,MicrobiologyandImmunology,VanderbiltUniversitywithLucVanKaerAdrianaZolini,PhD(Advisor:PJHansen)Currently,DairyBusiness,Zoetis,SiouxFalls,SD

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HISTORYOFTHEAMCBRESEARCHSYMPOSIUM

YEAR LOCATION DISTINGUISHEDLECTURER2003 WhitneyLaboratory

St.Augustine,FloridaRandyPrather

UniversityofMissouri2004 ChinsegutHill

Brooksville,FloridaJohnDobrinsky

USDA‐ARSBeltsville,MD2005 ChinsegutHill

Brooksville,FloridaDougStocco

TexasTechUniversity2006 LakeWauburg

Gainesville,FloridaInaDobrinski

UniversityofPennsylvania2007 WhitneyLaboratory

St.Augustine,FloridaDougBannerman

USDA‐ARS,Beltsville,MD2008 CedarCoveBeach&YachtClub

CedarKey,FloridaEckhardWolf

LMUMunich,Germany2009 PlantationGolfResortandSpa

CrystalRiver,FloridaDeanBetts

UniversityofWesternOntario2010 WhitneyLaboratory

St.Augustine,FloridaMarc‐AndreSirardLavalUniversity

2011 SteinhatcheeLandingResortSteinhatchee,Florida

KimberlyVonnahmeNorthDakotaStateUniv.

2012 HolidayIsleOceanfrontResortSt.Augustine,Florida

RocíoRiveraUniversityofMissouri

2013 HarborFrontHamptonInnFernandinaBeach,Florida

MartinSheldonSwanseaUniversity

2014 LakesideInnMountDora,Florida

CynthiaBaldwinUniversityofMassachusetts

2015 JekyllIslandClubHotelJekyllIsland,Georgia

PatLonergan&TrudeeFairUniversityCollegeDublin

2016 ChinsegutHillRetreatBrooksville,Florida

ShawnDonkinPurdueUniversity

2017 SafetyHarborResortandSpaSafetyHarbor,Florida

KieranMeadeTeagasc,Co.Meath,Ireland

2018 PlantationonCrystalRiverCrystalRiverFlorida

ChristopherGeyerEastCarolinaUniversity

2019 InternationalPalmsResort&Conf.Center,CocoaBeach,Florida

BrianO’NeillUniversityofIowa

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SCHEDULEOFEVENTS

AlleventsintheManateeRoomunlessotherwisenoted

FRIDAY,APRIL121:05PM PeteHansen

Welcome,introductorycomments

Session1:MercuryRoneyZimpelandChengYe,Chairs1:15PM WeihongGu,AnimalSciences

iNKTcells:Russianrouletteorasilverbulletininfluenza1:30PM JasonRizo,AnimalSciences

SeminalplasmaorTGFβmodulatesexpressionofproinflammatorycytokinesinbovineendometrialcells

1:45PM BoZhang,AnimalSciences

DetermininghowmuchATPisyieldedfromfermentationbyrumenmicrobes

2:00PM DarlingMelanydeCMadrid,AnimalSciencesTargetingNKTcellstoblockinfluenzaAinfectionsinswine

2:15PM EliabEstrada‐Cortes,AnimalSciences

Developmentalprogrammingofbovinepreimplantationembryosbycholinechloride

2:30PM BREAKSession2:2019AMCBDistinguishedLecturerPresentationJoséE.P.Santos,Chair3:00PM BrianT.O’Neill UniversityofIowaCarverCollegeofMedicine

Insulinsignalingintheregulationofmusclestrengthandmitochondrialmetabolism

4:00PM BREAKANDCHECKINTOROOMS

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Session3:GeminiAliHusnainandThiagoAmaral,Chairs

4:30PM RoneyZimpel,AnimalSciences

EffectsofdietaryvitaminD3or25‐hydroxyvitaminD3onmineralmetabolismingrowingcalves

4:45PM ChengYe,AnimalSciences

Unmaskingakiller:discoveryofagenethatcontrolsthepathogenicactivationofCD8TcellsinType1diabetes

5:00PM ZairaMagdalenaEstradaReyes,AnimalSciences

AssociationanalysisidentifiesTh1/Th2/ThreglociassociatedwithresistancetonaturalHaemonchuscontortusinfectionsinFloridaNativesheep

5:15PM SenaL.Field,AnimalSciencesSupplementationof5‐HTPorfluoxetineimpactsbioenergeticsindairycalves

5:30PM Break6:00PM Groupphotoonthebeach(entrancetobeachnexttoMambo’sGrill) 7:00PM SOCIALandDINNER,Mambo’sGrill(restaurantonsite)

SATURDAY,APRIL137:00‐8:30AM BREAKFAST,TheBreakfastSpot(restaurantonsite)Session4:ApolloMelanyDarlingMadridandandFroylanSosaHernandez,Chairs8:45AM YaoXiao,AnimalSciences

DickkopfWNTsignalingpathwayinhibitor1isnotabletomaintainpluripotencyduringderivationofbovineembryonicstemcells

9:00AM PeixinFan,AnimalSciencesandEmergingPathogensInst.

UnderstandingtheroleofhostgeneticsongutmicrobiotathroughlifebyusingtheAngus‐Brahmanmultibreedherd

9:15AM UsmanArshad,AnimalSciences

Meta‐analysisoftheeffectsofsupplementalrumen‐protectedcholineduringthetransitionperiodonperformanceandhealthofdairycows

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9:30AM SamanthaR.Bohm,AnimalSciences

Effectsofdietary25‐hydroxyvitaminD3onvitaminDstatusandpubertyofdairyheifers

9:45AM MichaelB.Poindexter,AnimalSciences

EffectsofdoseandsourceofvitaminDonmineralhomeostasisandperformanceintransitiondairycows

10:00AM PaulaC.C.Molinari,AnimalSciences

Dairycowshaveanincreasedincidenceofuterinediseaseduringthehotseasonwithnodifferenceinvaginalbacteriaload

10:15AM BREAKSession5:SpaceShuttleUsmanArshadandSenaField,Chairs10:30AM AchillesVieira‐Neto,AnimalSciences

EffectofdurationofexposuretodietsdifferinginDCADonCametabolismafteraparathyroidhormone(PTH)challengeindairycows

10:45AM FroylanSosaHernandez,AnimalSciencesEffectofcolonystimulatingfactor2oncompetenceofbovineblastocyststosurvivevitrification

11:00AM RachelL.Piersanti,AnimalSciences

Shortandlongtermeffectsofuterinediseaseonoocytetranscriptomeindairycows

11:15AM AliHusnain,AnimalSciences

Impactofinduceduterineinflammationonconceptusdevelopmentindairycows

11:30AM BREAKSession6:InternationalSpaceStationMichaelPoindexterandWeihongGu,Chairs11:45AM CourtneyMcCourt,AnimalSciences

Catalyticcharacterizationoftheenzymeinvolvedinthefinilstepofacetateformation:inSelenomonasruminantiumHD4

12:00PM MackenzieDickson,AnimalSciencesUterinediseaseimpairsbovineoocytequalityandembryodevelopmentinvitro

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12:15PM ThiagoAmaral,AnimalSciences

ExpressionofKREMEN1inthebovinepreimplantationembryo

12:30PM Closing(andverybrief)remarks,PeteHansen

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SitemapofInternationalPalmsCocoaBeachHotel&Report

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ABSTRACTS(Arrangedalphabeticallybyfirstauthor)

Expression of KREMEN1 in the bovine preimplantation embryo Thiago F. Amaral and Peter J. Hansen Department of Animal Sciences, University of Florida, Gainesville, Florida, USA Embryonic development is regulated by signaling molecules produced by the reproductive tract and embryo. Among these molecules, dickkopf-1 (DKK1) is a secreted inhibitor of canonical WNT signaling expressed in endometrium that can increase the competence of bovine blastocysts to establish pregnancy after transfer into recipients. The mechanism by which DKK1 increases embryo competence is not known. The long-term goal of this research is to identify cell signaling pathways through which DKK1 affects embryo function. Actions of DKK1 on other cells involve either inhibition of canonical WNT signaling or activation of the planar cell polarity (PCP) pathway. Inhibition of the canonical WNT pathway by DKK1 involves endocytosis of

LRP5/6 WNT co-receptor by formation of a ternary complex of DKK1/KREMEN/LRP. The current hypothesis being examined is that DKK1 regulates embryo function through actions involving

KREMEN-mediated internalization of LRP5/6. As a preliminary experiment, we evaluated expression of the two KREMEN genes (KREMEN1 and KREMEN2) in bovine embryos. First, data mining of an existing transcriptome dataset of morula, blastocyst

inner cell mass and blastocyst trophectoderm (BMC Dev Biol 12:33) was performed. Both KREMEN1 and KREMEN2 were expressed in all cell types examined, with transcript abundance of KREMEN1 being 5 to 14-fold greater than for KREMEN2. Highest expression of KREMEN1 was in morula (50 reads for morula, 11 reads for inner cell mass and 29 reads for trophectoderm). Subsequently, reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm expression of KREMEN1 by in vitro produced blastocysts using two sets of primers. Endometrium was used as a positive control. Results confirmed expression of KREMEN1 in both blastocysts and endometrium (Figure 1). Future experiments will be conducted to determine whether knocking down mRNA for KREMEN1 blocks effects of DKK1 to alter blastocyst gene expression and reduce accumulation of β-catenin.

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Meta-analysis of the effects of supplemental rumen-protected choline during the transition period on performance and health of parous dairy cows U. Arshad, M. G. Zenobi, C. R. Staples, and J.E.P. Santos

Department of Animal Sciences, University of Florida, Gainesville 32611

The objectives were to use meta-analytic methods to determine the effects of supplementing rumen-protected choline starting prepartum on production and health of parous dairy cows. The literature was systematically reviewed and 21 experiments with 66 treatment means, with a range of choline ion fed prepartum, from 3.2 to 25.2 g/d of diet, reported data for 1,309 parous cows. Duration of prepartum feeding of diets reported in the experiments averaged (± SD) 22.0 ± 5.9 days. All 21 experiments had a non-supplemented treatment (0 g choline/d). Data collected included number of cows, days on treatment pre- and postpartum, choline ion fed (g/d), ingredients and nutrient content of prepartum diets, number of days of measurements postpartum, and LSM and respective SEM for productive and health responses. The contents of diet NEL (Mcal/kg), metabolizable protein (MP) as % of DM intake (MPDMI), and metabolizable methionine as % of MP (METMP) were estimated for each prepartum diet using NRC (2001). Mixed models were fitted using the MIXED and GLIMMIX procedures of SAS. Models contained the random effect of experiment and data were weighted by the inverse of the SEM squared to account for the precision of each experiment. Models included the linear and quadratic effects of choline ion, diet NEL, MPDMI, and METMP, days on treatment prepartum, milking frequency, and interactions between choline and diet NEL, and choline and METMP in parous cows. Additional meta-analytical statistics were used to estimate the effect size using the METAN and METAREG procedures of STATA. The estimated effect of supplementing 0 vs. 12.9 g/d of choline ion during the transition period is presented in Table 1. Increasing prepartum intake of choline ion linearly (P<0.001) increased pre- (β = 0.018 ± 0.004; weighted mean difference [WMD] = 0.27 kg/d) and postpartum DM intake (β = 0.045 ± 0.012; WMD = 0.59 kg/d), energy-corrected milk (β = 0.174 ± 0.021; WMD = 1.90 kg/d), fat yield (β = 0.006 ± 0.001; WMD = 0.08 kg/d), and protein yield (β = 0.005 ± 0.001; WMD = 0.06 kg/d) in parous dairy cows. However, supplementing prepartum choline ion had no effect on retained placenta, mastitis, displaced abomasum, ketosis, or metritis in parous cows. Although, responses were linear up to 25 g/d of choline ion, yet, the dose of choline ion fed did not allow for the detection of an optimum value for postpartum performance.

Table 1. Effect of prepartum supplementation of 0 vs. 12.9 g of choline ion on performance (LSM ± SEM) in parous transition dairy cows Item Control Choline1 P-value Prepartum

DM intake, kg/d 12.0 ± 0.5 12.3 ± 0.5 < 0.001 Postpartum

DM intake, kg/d 20.5 ± 0.8 21.0 ± 0.8 < 0.001 ECM, kg/d 35.5 ± 1.6 37.7 ± 1.6 < 0.001 Fat yield, kg/d 1.30 ± 0.07 1.38 ± 0.07 < 0.001 Protein yield, kg/d 1.05 ± 0.04 1.12 ± 0.04 < 0.001

1 Choline = Linear effect of supplementation of choline ion during transition period

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Effects of dietary 25-hydroxyvitamin D3 on vitamin D status and puberty of dairy heifers S. R. Bohm, M. B. Poindexter, A. C. M. Silva, T. L. Williams, and C. D. Nelson Department of Animal Sciences, University of Florida, Gainesville, FL, USA Vitamin D plays a role in maintaining calcium and phosphorous homeostasis and is required for normal growth and development of heifers. We hypothesized that supplementing dairy heifers with 25-hydroxyvitamin D3 [25(OH)D3] would improve growth and development of dairy heifers. The objective of this experiment was to test the effects of dietary 25(OH)D3 on growth and the onset of puberty in dairy heifers. One hundred forty-three Holstein dairy heifers were assigned to one of two treatments, control (CON) and 25(OH)D3 (25D) starting at approximately 14 d of age. Control diets were formulated to provide vitamin D3 at a rate of 1.5 μg vitamin D3/kg BW and 25D heifers were supplemented with an additional 1.7 g 25(OH)D3/kg BW. Treatments were applied three times weekly to individual heifers. Analysis of growth data from birth to 3 m of age indicated 25D calves had greater BW gain through 3 m of age compared to the CON. For the present report serum 25(OH)D3 and BW were analyzed from 3 to 9 m of age. Age at onset of puberty was analyzed by concentrations of progesterone in biweekly serum samples collected from heifers when they were between 200 to 300 kg BW. Serum 25(OH)D3 and BW data were analyzed with the mixed procedure of SAS that included fixed effects of treatment and age and heifer within treatment as a random effect. Age and BW at onset of puberty was defined as serum progesterone greater than 1 ng/mL. Serum 25(OH)D3 concentrations of 25D heifers were greater (P < 0.001) compared with CON (118 vs. 59 ± 4 ng/mL). Effect of treatment (P = 0.03) and interaction between treatment and age (P = 0.05) were significant such that heifers receiving 25D were heavier at 3 m of age compared with CON heifers (109 vs. 113 kg) but BW of CON heifers increased over time such that at 9 m of age, BWs were similar between 25D and CON heifers (252 vs. 254 kg). Age and BW at puberty did not differ between treatment groups with BWs of (267 ± 5 kg) and age being (283 ± 6 d) at onset of puberty. In conclusion, these data suggest that the supplementation of 25(OH)D3 improves serum 25(OH)D3 levels. Although 25(OH)D3 supplementation increases BW up to 3 m of age, the difference in BW was not maintained and it had no effect at age at onset of puberty.

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Uterine disease impairs bovine oocyte quality and embryo development in vitro Mackenzie J. Dickson1, Jeremy C. Block2, José E. P. Santos1, I. Martin Sheldon3, and John J. Bromfield1

1Department of Animal Sciences, University of Florida, FL 32611. 2OvaTech, LLC., FL 32609. 3Institute of Life Science, Swansea University Medical School, Swansea, SA2 8PP, UK. Uterine disease is common in postpartum cows and is associated with subsequent subfertility even after resolution of disease. We used an experimental infection model of uterine disease to characterize the impact of bacterial endometritis on the oocyte and its ability to develop into an embryo in culture. Estrous cycles were synchronized in 23 non-lactating, primiparous Holstein cows, and cows were blocked by days postpartum then assigned randomly to receive an intrauterine infusion of vehicle (Luria-Bertani broth; Control, n=11), or endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; Bacteria, n=12) on day 2 of the estrous cycle. Data were analyzed by ANOVA for continuous data and logistic regression for binomial data. Increased endometrial expression of CXCL8, IL1B, IL6, PTGS2 and TNF on day 6 after infusion, and the presence of vaginal pus confirmed acute endometrial inflammation in bacteria infused cows compared with controls. Oocytes were collected 2, 24, 45, and 66 days after intrauterine infusion by transvaginal ultrasound guided oocyte pick-up and used for in vitro fertilization. The number of oocytes collected did not differ between treatments, but there was a tendency (P = 0.08) for a greater proportion of oocytes to undergo cleavage when collected from bacteria infused cows compared with controls. Interestingly, the proportion of cleaved zygotes to reach the morula stage of development was reduced (P = 0.04) when oocytes were collected from bacteria infused cows (33.9%) compared with controls (42.2%). Morulae derived from bacteria infused cows also had increased (P = 0.02) expression of heat shock 70 kDa protein 1A (HSPA1A) compared with controls. These data provide evidence that uterine disease reduces the capacity of oocytes to progress to the morula stage of development following in vitro fertilization. This work is supported by NICHD R01HD084316.  

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Developmental programming of bovine preimplantation embryos by choline chloride Eliab Estrada-Cortes1, William Ortiz1, Elizabeth Jannaman1, Charles R. Staples1, Jeremy Block2, and Peter J. Hansen1

1Department of Animal Sciences, University of Florida, Gainesville, Florida, USA. 2Ovatech, Gainesville, Florida, USA. The process of global DNA demethylation and re-methylation occur during the pre-implantation period in bovine embryos; therefore, this is a critical period to establish the epigenome. Choline is a precursor of betaine, a methyl donor for DNA methylation and supplementation with rumen-protected choline has improved reproductive performance in dairy cows. The objective was to determine whether addition of choline chloride to culture medium improves the competence of in vitro-produced embryos to become blastocysts, to establish pregnancy after embryo transfer (ET) and alter characteristics of resultant offspring. Oocytes were obtained by ovum pick up from Brahman cows (n=11). Oocytes from each donor were fertilized using conventional (n=4) or sexed semen (n=1) from five Brahman sires. Half of the presumptive embryos from each donor were incubated in BBH7 culture medium (control; n=387 oocytes) and half with culture medium containing 1.8 mM choline chloride (n=332 oocytes). Treatments were isotonic. At day 7, blastocysts were transferred into Angus x Brangus recipients. Pregnancy diagnosis was performed at day 28 after ET and resultant offspring were evaluated at birth. There was no effect of treatment (P>0.1) on the percent of cleaved embryos becoming blastocyst (15.9±3.4% control vs 15.8±3.3% choline) or pregnancy per ET [50.0% (31/62) vs 40.0% (20/50)] for control or choline, respectively. Birth weight was affected by the treatment x sex of calf interaction (P=0.0022). For females, choline calves were heavier than control calves [49.4±2.5 (n=4) vs 34.3±2.1kg (n=7)] whereas the opposite was observed for male calves [27.5±3.2 (n=3) vs 36.2±3.2kg (n=4)]. In conclusion, addition of 1.8 mM choline chloride to culture medium did not affect competence of embryos to become blastocysts but programmed development to alter fetal growth in a sex-dependent manner (Support: Larson Endowment).

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Association analysis identifies Th1/ Th2/ Threg loci associated with resistance to natural Haemonchus contortus infections in Florida Native sheep Zaira Magdalena Estrada Reyes, Owen Rae, Carol Postley, Myriam Berenice Jiménez Medrano, Joel Leal Gutiérrez, and Raluca Mateescu *Department of Animal Sciences, University of Florida, Gainesville, FL

The aim of this study was to identify single nucleotide polymorphisms (SNPs) associated to internal parasite resistance in Florida Native sheep, using a targeted sequencing approach. One hundred and fifty three lambs (5 months and three months old) were evaluated in this study. At the start of the trial, phenotypic records for fecal egg count (FEC), FAMACHA score, body condition score (BCS), and weight were recorded and deworming of sheep with levamisole (18 mg per kg of body weight) was performed. Ten and 28 days post deworming, a full hematogram of each sheep was obtained and FEC, FAMACHA score, BCS and weight were assessed. Average daily gain was calculated at the end of the trial. Out of 153 animals, 100 sheep were selected for genotyping using a targeted sequencing approach. Targeted sequencing panel included 100 candidate genes for immune response against H. contortus. SNPs were discarded if call rate < 95% and minor allele frequency ≤0.05. A mixed model was used to analyze the response variables and included the identity by state matrix to control for population structure. A contemporary group (age, group and sex) was included as fixed effect. Bonferroni correction was used to control for multiple testing. Eighteen SNPs on chromosomes 1, 2, 3, 4, 6, 7, 11, 15, 18, 20 and 26 were significant for different traits. For FEC, significant SNPs were located within ITGA4, STAT3, MUC15, IL2RB and CFI genes. Two SNPs within IL12RB2 and GPX2 genes were significantly associated with FAMACHA score. One SNP in IL16 gene was associated with both red blood cell count (RBC) and hemoglobin level (HGB). Significant SNPs in STAT6, PCDH7 and MUC15 genes were identified for white blood cell count (WBC). CXCL10, TNF, CCL26, ITGA4 and TLR3 genes contained significant SNPs associated with neutrophil, basophil and eosinophil counts. These results suggest that loci related to Th1/Th2 and Treg responses play an important role in the expression of resistant phenotypes. In conclusion, potential immune like loci could be used as genetic markers for resistance in sheep exposed to natural H. contortus infections.

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Understanding the role of host genetics on gut microbiota through life by using the Angus-Brahman multibreed herd Peixin Fan1, 2, C. D. Nelson1, J. D. Driver1, M. A. Elzo1, and F. Peñagaricano1, K . C. Jeong1, 2*

1Department of Animal Sciences, University of Florida, Gainesville, FL

2Emerging Pathogens Institute, University of Florida, Gainesville, FL  

Commensal bacteria in the intestinal tract provide energy for the host and inhibit pathogen colonization. However, the role of host genetics on the gut microbiota structure and its consequences on animal growth are largely unknown. In this study, we investigated the effects of breed composition on the gut microbiota of Angus-Brahman multibreed (MAB) calves at three different growing stages. Fecal samples were collected from a cohort of MAB calves at preweaning, postweaning and fattening stages, and gut microbiota structure was analyzed by using 16S rRNA gene sequencing. We found that breed composition influenced the gut microbiota structure at all three stages, but the variation in the relative abundance of bacteria driven by breed composition was greater in the preweaning and fattening stages compared to the postweaning stage. The relative abundance of Clostridium and unclassified Ruminococcaceae as well as microbial genes involved in nitrogen metabolism showed consistent positive correlation with Angus proportion through life, while the relative abundance of Sutterella was positively correlated with Brahman proportion. Interestingly, weight gain of the MAB calves was higher in calves with more Angus proportion in the preweaning and fattening stages, but did not show significant difference in the postweaning stage. Strong positive correlations were detected between weight gain and the relative abundance of butyrate producing bacteria Faecalibacterium, Blautia, Oscillospira, and Prevotella only at the preweaning stage, suggesting profound effect of preweaning gut microbiota on animal growth. Moreover, SNP genotyping showed high agreement between breed composition estimated from pedigree and genetic distance, and 87,807 SNP markers showed correlation with breed composition. Further studies will identify the relationship between host SNPs and abundance of bacteria to understand the role of genetic effects on the gut microbiota. In conclusion, breed composition influences the gut microbiota of MAB calves greatly depending on growing stages that contributes to animal growth.

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Supplementation of 5-HTP or fluoxetine impacts bioenergetics in dairy calves Field S.L.*, Marrero M.G., Skibiel A.L., Dado-Senn B. and J. Laporta Department of Animal Sciences, University of Florida, Gainesville, 32611 Serotonin (5-HT) is a monoamine that regulates energy balance through the modulation of insulin and lipid metabolism. Here, we examined if manipulating 5-HT pathway by administering Fluoxetine (FLX, a 5-HT reuptake inhibitor) or 5-hydroxytryptophan (5-HTP, a 5-HT precursor), would impact energy metabolism in pre-weaned dairy calves. Bull Holstein calves (212 d) were fed milk replacer (8 L/d) with saline (CON, 8 mL/d n=8), FLX (40 mg/d, n=8) or 5-HTP (90 mg/d, n=8) for 10 consecutive d in a complete randomized block design. Blood samples were collected before (d-1), during treatment (d1-10) and during withdrawal (d2, 3, 4, 7, 14) periods to measure insulin and NEFA concentrations. Calves were euthanized after treatment or withdrawal period to harvest pancreas and adipose tissue to measure gene expression of 5-HT receptors (5-HTR), 5-HT transporter (SERT) and tryptophan hydroxylase (TPH1) by real-time PCR. Data was analyzed by period using one and two-way ANOVAs in R. Insulin had a treatment by day interaction (P<0.01), where 5-HTP group had higher circulating concentrations compared to the CON, but had only a day effect during withdrawal period (P<0.001). Circulating NEFA concentrations were not different during treatment or withdrawal period (P>0.10). In the adipose and pancreas, for both treatment and withdrawal periods, SERT, TPH1 and 5-HTR were expressed with the exception of 5-HT1D, -2C, -3A, -3B and -4, whereas 5-HT5 was only expressed in pancreatic tissue and 5-HT6 in adipose tissue. After treatment period, adipose gene expression of 5-HT1A tended to be downregulated in the 5-HTP group (P<0.10), but after withdrawal period 5-HT1F was upregulated by 5-HTP and FLX (P<0.01) when compared to CON. After treatment period, there was a tendency for downregulation of pancreatic gene expression of SERT (P<0.08) and 5-HT1F (P<0.10), in FLX and 5-HTP group, respectively, when compared to CON. After withdrawal, serotonin pancreatic receptors were not differentially expressed. Manipulating serotonin bioavailability of dairy calves increases circulating insulin and modifies the expression of serotonin receptors in the pancreas and adipose tissue.

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iNKT cells: Russian roulette or a silver bullet in influenza A virus immunity Weihong Gu, Darling Melany de C. Madrid, Guan Yang, Bianca L. Artiaga and John P. Driver Department of Animal Sciences, University of Florida, Gainesville, FL, 32611 CD1d-restricted invariant natural killer T (iNKT) cells are a distinct subset of T cells that express NK cell receptors. iNKT cells are called “bridging cells” because they have characteristics of both the innate and adaptive immune system. Not all mammals express iNKT-CD1d system, but pigs do. We have found that pigs are more similar to humans than mice for the frequency, distribution, NKT cell T cell receptor structure and antigen recognition. Along with their similar size to humans, these qualities make swine an attractive preclinical model to study the physiological role of iNKT cells for humans. There is also value in understanding how iNKT cells contribute to immunity against important pig pathogens. However, current understanding of porcine iNKT cells is rudimentary, and in need of study. Because single-cell RNA sequencing is a powerful tool to distinguish rare immune cell subsets and dynamic cellular changes at transcriptomic level, we are using this technology to establish the developmental pathway of iNKT cells in pig thymus and determine functional iNKT subsets. Here, we report a magnetic bead-based purification technique that we developed to enrich thymic iNKT cells for sequencing. Also described is a recent study to study the contribution of porcine iNKT cells to immunity against influenza A virus (IAV), a pathogen of great importance for humans and swine. In contrast to what has been reported for mice, it was found that iNKT cell-deficient pigs have reduced and delayed virus shedding compared to iNKT cell intact pigs, indicating that iNKT cells may induce inflammatory immune responses that actually hinder virus clearance. This implies that pigs and humans with high iNKT cell levels could be more at risk for spreading influenza during an outbreak.

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Impact of induced uterine inflammation on conceptus development in dairy cows

A. Husnain, John Bromfield, and J.E.P. Santos

Department of Animal Sciences, University of Florida, Gainesville FL 32611

Epidemiological data in dairy cows demonstrate that postpartum uterine diseases, which affect 20 to 40% of postpartum cows, are associated with reduced pregnancy per artificial insemination, and increased risk of pregnancy loss. Despite the extensive body of work, the underlying mechanisms resulting in failure of establishment and maintenance of pregnancy remain mostly unclear. We hypothesize that inflammation of the uterus alters the uterine environment that impairs conceptus development in dairy cows. The aim of the current experiment is to utilize an animal model of induced endometritis to investigate the underlying molecular mechanisms of how uterine inflammation affects conceptus development during the peri-implantation period. Postpartum lactating dairy cows with no history of retained placenta, metritis, or clinical endometritis and having metricheck score < 2 and endometrial cytology with less than 10% of polymorphonuclear neutrophils on 23 ± 3 days in milk (DIM), will be enrolled. Cows will have their estrous cycle synchronized using the double Ovsynch protocol starting at 25 DIM. Cows will be assigned randomly to receive no intrauterine treatment (negative control, NC; n = 18) or to be treated (n =18) at 40 DIM with an intrauterine inoculation of 5 x 108 CFU of Escherichia coli and 5 x 108 CFU Trueperella pyogenes, a model already validated to induce inflammation and signs compatible with clinical endometritis. The uterus of cows will then be evaluated for discharge, presence of endometrial polymorphonuclear neutrophils and expression of pro-inflammatory cytokines (IL-1, IL-6, IL-12, TNF and IFNγ). Single fresh in vitro produced Holstein embryo will be transferred on day 7 of the estrous cycle (56 DIM) and the uteri will be flushed 9 days later, on day 16 of the presumptive pregnancy. The uterine flush will be collected and frozen for later quantification of interferon-tau, pro-inflammatory markers, and characterization of the lipidome and metabolome through mass-spectrometry. After flushing, endometrial tissue from the horn ipsilateral to the corpus luteum will be collected for biopsy for fatty acid analysis and quantification of mRNA expression for genes of interest. Immune cell infiltration into the endometrial tissue will be characterized through immunohistochemistry to identify uterine natural killer cells, macrophages and T- lymphocytes. The length of recovered concepti will be measured and the presence of the embryonic disc identified. Conceptus mRNA expression will be investigated to characterize genes involved in fatty acid metabolism, cell signaling, growth and development, inflammation, immune function, cell division, and apoptosis through real time PCR in fluidigm platform (Biomark HD). Blood will be collected throughout the experiment for measurements of concentrations of progesterone, haptoglobin, serum amyloid A, and total and differential white blood cell count. Concentrations of estradiol will be measured during proestrus immediately before induction of ovulation. It is anticipated that inducing endometritis will increase plasma and endometrial markers of inflammatory response. It is expected that elevated pro-inflammatory cytokine would upregulate the expression of non-classic major histocompatibility complex class I (MHC-1) on trophoblast and activate the genes associated with cell death and/or apoptosis in conceptus. Infiltration of immune reactive cells into the uterus and their interactions with the upregulated MHC-1 on trophoblast would initiate the immune response that potentially result in death of the embryo. In addition, it is speculated that altered histotroph composition most likely affects the metabolism of fatty acids and glucose, mediated through peroxisome proliferator activated receptor gamma (PPARγ), resulting in reduced embryo elongation that eventually will lead to less production of IFN-tau.

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Targeting NKT cells to block influenza A infections in swine

Darling Melany de C. Madrid, Weihong Gu, John P. Driver

Department of Animal Sciences, University of Florida, Gainesville. Antiviral therapy options for treatment of influenza A(IAV) infection are limited for humans and animals. The leading therapy currently used to treat influenza in humans, oseltamivir (Tamiflu®), has to be administered at early onset of symptoms to affect the course of disease, which requires quick diagnosis of symptoms. In addition, cases of viral resistance against this drug have been reported because the therapy targets the virus. Thus, there is a need for novel antiviral therapies that can overcome these limitations by targeting the immune system. Swine are natural hosts for influenza A virus and are also capable of being infected by virus of human and avian origin, which makes them a potential “mixing vessel” for reassortant virus. Currently, there is no antiviral available to control disease pathology and transmission for commercial swine. It has been reported in mice that the activation of natural killer T cells (NKT), a special subtype of T cell, can improve the outcome of diseases caused by a plethora of pathogens, including influenza A virus. Studies show that NKT cell activation reduces mortality and viral shedding in IAV-infected mice. Pigs and humans also possess this special population of cells that can be activated by a synthetic marine-sponge-derived agonist, α-Galactosylceramide (α-GalCer). We describe how intranasal delivery of α-GalCer to pigs infected with IAV inhibited viral replication in airway tissues, decreased virus shedding and also reduced lung pathology without causing any disadvantageous immune reaction in newly-weaned pigs. We also show that NKT cell agonists therapy is comparable to oseltamivir for controlling lung pathology and virus shedding in young piglets infected with pandemic H1N1influenza virus A/CA/04/09. Our results demonstrate the potential to use NKT cell therapy to block influenza transmission in swine and possibly humans, which is needed and deserves further study.

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Catalytic characterization of the enzyme involved in the finil step of acetate formation: in Selenomonas ruminantium HD4 C. McCourt, B. Zhang, C. Brown, and T. Hackmann Department of Animal Sciences, University of Florida, Gainesville The majority of bacteria in the ruminant are unculturable, and their substrate consumption as well as product formation must be predicted from reconstructed genomes. The prediction method most commonly used only includes the classic pathways and their respective enzymes. Selenomonas ruminantium HD4 a well characterized rumen bacterium was used to validate this method of pathway prediction. The results of which reveal that S. ruminantum HD4 is not predicted to produce acetate, yet S. ruminantum HD4 has been experimentally validated to produced acetate through Acetate Thiokinase. Our hypothesis is to validate an alternative pathway predicted by a less exclusive method to which Succinyl:Acetyl-CoA Transferase is used in the conversion of acetyl-coa to acetate and not Acetate Thiokinase, in S. ruminantium HD4. Extracts from S. ruminantum HD4 were tested for the enzymatic activity of Acetate Thiokinase, and Succinyl:Acetyl-CoA Transferase. Additionally, extracts from S. ruminantum HD4 were dosed with cofactors predicted in the enzymatic reaction to secondarily validate results of enzymatic activity. Methods where completed aerobically as well as anaerobically. We detected no enzymatic activity for Acetate Thiokinase, and Succinyl:Acetyl-CoA Transferase in S. ruminantum HD4.We did detect that phosphate significantly increased (P<0.05) the ability of the unknown enzyme to catalyze the reaction from acetyl-coa to acetate. The addition other cofactors had no effect: pyrophosphate, AMP and pyrophosphate, ADP and phosphate, GDP and phosphate, Succinate and phosphate, Succinate ADP and phosphate, or Succinate GDP and phosphate. We further determine that the unknown enzyme was not aerotolerant, requiring anaerobic conditions. We reject our hypothesis that Succinyl:Acetyl-CoA Transferase is used in the conversion of acetyl-coa to acetate, as well we further provide evidence against Acetate Thiokinase. We conclude by hypothesizing that a hydrolase is the unknown enzyme responsible for the conversion of acetyl-coA to acetate.

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Dairy cows have an increased incidence of uterine disease during the hot season with no difference in vaginal bacteria load

Paula C.C. Molinari1, I. Martin Sheldon2 and John J. Bromfield1.

1Department of Animal Sciences, University of Florida, Gainesville FL 32611. 2Institute of Life Science, Swansea University Medical School, Swansea, SA2 8PP, UK

Bacterial infections cause metritis about 7 days after calving in approximately 30% of dairy cows, and uterine disease persists as endometritis in some animals beyond 21 days postpartum. Although heat stress reduces milk production and impairs immune function, little is known about its effect on uterine infection and disease. Our aim was to evaluate uterine disease severity and quantify the bacterial load in vaginal mucus collected from dairy cows on the same farm, during a cool (n=51, average maximum temperature: 22.5°C) season and a hot season when animals are subjected to heat stress (n=51, 31°C). We hypothesize that the incidence and severity of uterine disease is increased during the hot season, accompanied by an elevated bacterial load in the reproductive tract. All animals received heat abatement with fans and sprinklers after calving. Vaginal mucus was scored according to the abundance of purulent discharge and odor on days 7 and 21 postpartum, at the time of diagnosis of metritis and endometritis, respectively. Bacterial 16S rRNA was quantified by qPCR in vaginal mucus samples. During the hot season a higher proportion of cows had persistent uterine disease at both day 7 and day 21 (58.8% vs. 29.4%) and increased incidence of endometritis (64.7% vs. 43.1%). The concentration of bacterial 16S rRNA in vaginal mucus was higher in animals with endometritis compared with healthy cows at day 21 (2.35 vs. 0.04 ng/mg mucus) but did not differ between the hot and cool seasons (2.12 vs. 2.68 ng/mg mucus). In conclusion, our data indicate that despite a similar vaginal bacteria load, heat stress increased the incidence of endometritis. This work is supported by NICHD R01HD084316.

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Short and long term effects of uterine disease on oocyte transcriptome in dairy cows. Rachel L. Piersanti1, Jeremy Block2, Jose E. P. Santos1, I. Martin Sheldon3 and John J. Bromfield1. 1Department of Animal Sciences, University of Florida, Gainesville, FL, USA. 2OvaTech, Gainesville, FL, USA. 3Institute of Life Science, Swansea University Medical School, Swansea, UK. Postpartum uterine disease is a common cause of reduced fertility in dairy cows. The effect of uterine disease on subsequent fertility may be related to the accumulation of pathogen-associated molecules and inflammatory mediators in follicular fluid, and their impact on oocyte development. In order to better understand the influence of uterine disease on the oocyte, we used an experimental model of induced uterine disease in virgin heifers. Animals received an intrauterine infusion of sterile medium (control, n=5) or bacteria (pathogenic Escherichia coli and Trueperella pyogenes, n=4). Uterine disease was confirmed in bacteria infused animals by the presence of clinical endometritis symptoms. Ovum pick-up was performed on day 4 (during disease) and day 60 (after resolution of clinical disease) relative to infusion, and zona-free oocytes were subjected to RNAseq analysis. Using a P-value < 0.05 cut-off, 474 genes were differentially expressed in oocytes from bacteria infused animals on day 4, and 929 genes on day 60. Only 56 genes were differentially expressed in oocytes of bacteria infused animals on both day 4 and day 60. Further analysis of the differentially expressed genes identified canonical pathways impacted at day 4 including, interferon signaling, TGFβ signaling, TNFR2 signaling, IL-6 signaling and BMP signaling; while at day 60 glycolysis, bile acid biosynthesis neutral pathway, gluconeogenesis, ILK signaling, interferon signaling and chondroitin sulfate degradation pathways were affected. Predicted upstream regulators of differentially expressed genes in oocytes of bacteria infused animals on day 4 included SOX2, LPS and NFκB; while upstream regulators at day 60 included IL-1β, IL-6, and LH. These data provide evidence that uterine disease alters the oocyte transcriptome, and that unique effects are also apparent in the oocyte after the resolution of disease. This work is supported by NICHD R01HD084316.

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Effects of dose and source of vitamin D on mineral homeostasis and performance in transition dairy cows

M.B. Poindexter1, A. Vieira-Neto1, A. Husnain1, R. Zimpel1, A. Faccenda1,2, A. Sanches de Avila1,2, A. Silva1, P. Celi3, C. Cortinhas3, J.E.P. Santos1, and C.D. Nelson1

1University of Florida, Gainesville, FL, USA, 2State University of Western Paraná, Cascavel, Paraná, Brazil, 3DSM Nutritional Products, Columbia, MD.

Objectives of the experiment were to determine the effects of supplementing two doses of either vitamin D3 (VitD) or 25-hydroxyvitamin D3 (25D) on peripartum mineral metabolism and lactation performance. One hundred and thirty-three parous and forty-four nulliparous pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield or genetic merit and then assigned randomly to receive a daily dietary top-dressed supplement containing 1 mg VitD (1mgD), 1 mg 25D (1mg25D), 3 mg VitD (3mgD), or 3 mg 25D (3mg25D) from 250 days in gestation until parturition. The prepartum diet was approximately -130 mEq/kg in dietary cation anion difference. Data were analyzed by ANOVA with mixed models using the Mixed procedure of SAS. Cows receiving the 25D treatments had greater (P = 0.01) concentrations of P in serum prepartum and a tendency for greater (P = 0.07) concentrations of P postpartum. Cows receiving the 25D treatments had greater (P = 0.04) concentrations of total Ca in serum postpartum. Cows receiving the 3mg25D treatment produced more milk (P < 0.01) in the first 42 d in milk compared with other treatments. Prepartum DM intake, postpartum disease incidence, colostrum production, and concentrations of Ca and Mg in colostrum and milk did not differ among treatments. Cows receiving 25D were in a better mineral status postpartum and performed better compared with cows receiving just VitD.

Item 1mgD 3mgD 1mg25D 3mg25D SE Prepartum Ca, mM 2.34 2.32 2.34 2.35 0.02 Mg, mM 0.85 0.84 0.83 0.83 0.02 P, mM* 1.89 1.87 1.96 2.05 0.03 Postpartum Ca, mM* 2.13 2.11 2.15 2.18 0.02 Mg, mM 0.88 0.85 0.87 0.84 0.02 P, mM 1.72 1.7 1.75 1.8 0.04 Colostrum yield, kg 4.3 3.9 5.4 4.5 0.55 Milk yield, kg/d¥ 35.3 32.4 33.4 36.6 1.3 *Source (P<0.05), §Level (P<0.05), ¥Level x Source (P<0.05)

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Seminal plasma or TGFβ modulates expression of proinflammatory cytokines in bovine endometrial cells

Jason A. Rizo and John J. Bromfield

Department of Animal Sciences, University of Florida, Gainesville FL 32611

Seminal plasma is the cell-free fraction of semen that induces endometrial inflammation following coitus in rodents and swine. Seminal plasma-derived TGFβ is the active molecule that modulates inflammation in female reproductive tissues. In rodents and swine, endometrial responses to seminal plasma promote embryo development and improve pregnancy outcomes. However, the endometrial response to seminal plasma is not well-characterized in cattle, where diluted semen is routinely used for artificial insemination. We hypothesize that seminal plasma or TGFβ increases expression of proinflammatory cytokines in bovine endometrial cells. Bovine endometrial (BEND) cells were treated for 24 hours with increasing concentrations of pooled seminal plasma (0.001% to 20%), rhTGFβ-1 (1 to 100 ng/mL) or rhTGFβ-2 (0.1 to 10 ng/mL). Cell viability was measured by MTT assay and gene expression was quantified by qPCR. Viability of BEND cells was reduced following exposure to 1% seminal plasma and higher concentrations. Heat-treatment of seminal plasma reduced cytotoxic effects of seminal plasma. Exposure of BEND cells to 1% seminal plasma (non-cytotoxic) increased expression of IL1B and IL6 compared to controls (6.7-fold and 36.7-fold), while expression of TNF and CSF2 was unchanged. Exposure of BEND cells to rhTGFβ-1 or rhTGFβ-2 increased TNF expression in the absence of estrogen, while IL6 expression was increased only after estrogen supplementation. Exposure to TGFβ did not affect CSF2 or IL1B expression. These data suggest that seminal plasma is cytotoxic to BEND cells and alters expression of proinflammatory cytokines. Similar expression of cytokines were observed in BEND cells following TGFβ exposure. Further investigation is required to determine the extent by which semen components elicit endometrial inflammation in the bovine which may be useful to optimize reproductive efficiency. This research was supported by Select Sires and the Southeast Milk checkoff.

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Effect of colony stimulating factor 2 on competence of bovine blastocysts to survive vitrification

Sosa, F. and P.J. Hansen

Department of Animal Sciences, University of Florida, Gainesville, Florida, USA.

Colony stimulating factor 2 (CSF2) is an important regulator of embryonic development. Treatment of in vitro produced embryos with CSF2 from days 5 to 7 after insemination can increase competence of embryos to establish pregnancy after transfer. One action of CSF2 is to protect embryos from stress. Treatment with CSF2 reduced heat-shocked induced apoptosis of blastomeres and increased the percent of embryos developing to the blastocyst stage after heat shock. Here we tested whether CSF2 would promote survival of embryos to another stress: cryodamage caused by vitrification. Ovaries were recovered from an abattoir and used to harvest cumulus-oocyte complexes (COC). Groups of 10 COC were matured for 20-22 h. After maturation, COC were fertilized with ̴1x106/mL Isolate®-purified spermatozoa from a pool of frozen-thawed semen from 3 bulls. Fertilization proceeded for 20-22 h. Cumulus cells were removed from putative zygotes using 1000 U/ml hyaluronidase by agitation and putative zygotes were cultured in groups of 30 in 50-µL drops of synthetic oviductal fluid – bovine embryo 2 (SOF-BE2) medium until day 7 after insemination. Drops of cultured embryos were randomly assigned to receive either 10 ng/ml CSF2 or vehicle [90% (v/v) SOF-BE2 and 10% (v/v) Dulbecco’s phosphate buffered saline containing 1 mg/ml bovine serum albumin) on day 5. Blastocyst stage embryos were selected for vitrification at Day 7. Blastocysts were vitrified and rewarmed according to procedures of (Vajta et al. Theriogenology 45: 683-689, 1996). After warming, embryos cultured for 72 h in groups of 5-12 in SOF-BE2 containing 10% fetal bovine serum and 50 µM dithiothreitol (DTT) were assessed for re-expansion (defined as restoration of a reconstituted blastocoele) and hatching (defined as partial or completion exit from the zona pellucida). To date, a total of eight replicates have been performed using 170 blastocysts treated with CSF2 and 185 blastocysts treated with vehicle. Data on percent re-expansion and hatching were calculated for each replicate and analyzed by analysis of variance using the GLM procedure of SAS. There were no significant differences due to CSF2 treatment (Table 1). Preliminary conclusions are that CSF2 does not increase blastocyst survival to vitrification. More replicates will be performed. In addition, vitrified blastocysts will be transferred to recipients to determine survival in vivo.

Re-expansion (%) Hatching (%) Treatment 24 h 48 h 72 h 24 h 48 h 72 h Vehicle 70 + 5 66 ± 5 66 ± 5 19 ± 4 32 ± 5 38 ± 4 CSF2 73 + 5 71 ± 5 71 ± 5 25 ± 4 32 ± 5 40 ± 4

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Effect of duration of exposure to diets differing in DCAD on Ca metabolism after a parathyroid hormone (PTH) challenge in dairy cows A. Vieira-Neto, I. M. R. Leão, J. G. Prim, K. V. Almeida, R. Zimpel, M. M. Nehme, J. Bollatti, A. C. M. Silva, A. R. Ruiz, C. D. Nelson, and J. E. P. Santos Department of Animal Sciences, University of Florida, Gainesville, FL, USA Objectives were to determine the length of exposure to an acidogenic diet (ACD) to elicit increased response to PTH-induced changes in blood Ca and vitamin D3 in prepartum cows. The hypothesis was that cows have increased PTH responsiveness within 3 d of feeding ACD. Ten parous Holstein cows at 2427 d of gestation were blocked by lactation and pretreatment DMI and, within block, they were assigned randomly to an alkalogenic (ALKD; DCAD = +209 mEq/kg DM; n=5) or an ACD (DCAD = -168 mEq/kg DM; n=5) on experiment d 0. Water and DMI were measured, and urine and blood were sampled. The PTH challenges were performed on d 3, 8, and 13. Cows received 0.05 g PTH/kg BW i.v. every 20 min for 9 h to mimic the pulsatile release of endogenous PTH. Jugular blood was sampled at 0 h, and hourly thereafter until 10 h, and at 12, 18, 24, 36, and 48 h relative to the challenge. Blood acid-base measures and concentrations of ionized Ca (iCa) were evaluated, and vitamin D metabolites will be assayed. Results were available for the first challenge on d 3 and data were analyzed by ANOVA with mixed models with SAS. Cows fed ACD had smaller (P<0.01) blood pH (7.429 vs. 7.382 0.005), base excess (4.3 vs. -2.4 0.5 mM), and bicarbonate (28.5 vs. 22.8 0.4 mM) within 24 h of the experiment compared with cows fed ALKD. Urine pH declined and differed (P<0.01) by 15 h of feeding the ACD (8.18 vs. 7.32 ± 0.17), and differences increased by 24 h (8.10 vs. 6.46 ± 0.17). Blood iCa increased (P<0.01) in ACD compared with ALKD by d 3 (1.216 vs. 1.281 0.013 mM). During the PTH challenge on d 3, cows fed ACD had greater (P<0.01) concentration of blood iCa than cows fed ALKD (1.331 vs. 1.423 0.013 mM). Nevertheless, the increment in iCa in the first 36 h after the challenge, relative to baseline at 0 h, did not differ between treatments (ALKD = 0.15 vs. ACD = 0.16 0.01 mM). Diet-induced metabolic acidosis occurred within 24 h of treatment; however, an increase in blood iCa concentration was observed after 3 d of metabolic acidosis. Blood iCa response to a PTH challenge did not differ between treatments on experiment d 3.

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Dickkopf WNT signaling pathway inhibitor 1 is not able to maintain pluripotency during derivation of bovine embryonic stem cells Yao Xiao1, Paula Tribulo1, Kenneth E. Diffenderfer2, Pablo J. Ross3 and Peter J. Hansen1

1Department of Animal Sciences, University of Florida, Gainesville, Florida, USA 2Stem Cell Core, Salk Institute for Biological Studies, La Jolla, California, USA 3Department of Animal Science, University of California, Davis, California, USA Pluripotent bovine embryonic stem cells (ESC) were recently derived from blastocysts by employing fibroblast growth factor 2 and a canonical WNT/β-catenin pathway inhibitor (IWR1) in a serum-free culture system. It suggested that modulation of the canonical WNT pathway played a critical role in establishment of pluripotency in bovine cells. Dickkopf WNT signaling pathway inhibitor 1 (DKK1) is a canonical WNT pathway inhibitor that controls lineage differentiation during embryogenesis. We hypothesis that IWR1 can be replaced by DKK1 during the derivation of bovine ESC. In vitro produced blastocysts (non-expanded, expanded or hatching) were treated to remove the zona pellucida, randomly divided into three groups and plated onto mouse embryonic fibroblast feeders with the standard ESC medium containing IWR1, DKK1 or vehicle (n=12 per group) in 4-well plates. Outgrowths were passaged 4-5 times and evaluated for immunolabeling with the pluripotency marker SOX2 at passages 3-5. A total of five embryo-derived cell lines were obtained from IWR1-treated cultures and all cell lines were positive for SOX2 at passage 4. Four cell lines were obtained from DKK1-treated cultures. Only one of these cell lines was positive for SOX2 at passage 3 and expression was gradually lost during subsequent passages. Four cell lines were derived from vehicle-treated cells and all were negative for SOX2. At passage 4, membrane-associated β-catenin (downstream signaling molecule for WNT signaling) was abundantly distributed on the cell membrane of IWR1-treated cells but was less abundant for both DKK1- and vehicle-treated cells. These data suggest that DKK1 was not as effective as IWR1 in maintaining pluripotency of embryo-derived cells. Perhaps, membrane-associated β-catenin has an important role in maintenance of pluripotency of cells derived from the bovine epiblast during culture. Future experiments will focusing on determining the lineage characteristics of DKK1-treated cells and testing whether IWR1 maintains pluripotency via stabilizing β-catenin and E-cadherin complexes in the plasma membrane to regulate cytoskeleton and cell adhesion.

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Unmasking a killer: discovery of a gene that controls the pathogenic activation of CD8 T cells in Type 1 diabetes Cheng Ye1, David Serreze2 Yi-Guang Chen3 and John Driver1

1Department of Animal Sciences, University of Florida, Gainesville; 2The Jackson Laboratory, Bar Harbor, ME; 3Department of Pediatrics, Medical College of Wisconsin, Milwaukee CD4 T cell help is critical for the activation of autoreactive CD8 T cells that cause most -cell destruction in type-1 diabetes (T1D). We previously identified a 10 Mb C57BL/6 (B6)-origin recessive Idd susceptibility locus on chromosome 11, designated Idd32 that controls whether CD4 T cells cause pathogenic CD8 T cells to undergo activation or become tolerized. To identify the underlying gene, we adoptively transferred CD8 T cells transgenically expressing the diabetogenic AI4 T cell receptor into B6 mice expressing the NOD-derived H2g7 MHC haplotype (B6.H2g7) and one of 9 different NOD-derived segments of the Idd32 interval. This reduced the interval to a <520 kb region containing 18 polymorphic genes. To test these candidate genes () we used the CRISPR/Cas9 system to generate two sets of F1 strains: (B6.H2g7.Idd32NOD/NOD.-/- x B6.H2g7)F1 and (B6.H2g7.Idd32NOD/NOD x B6.H2g7)F1 that were monitored for T1D development after infusing them with AI4 T cells. We found that F1mice lacking the NOD allele of the sialic acid transferase gene St6galnac1 (B6.H2g7.Idd32NOD/NOD. St6galnac1-/- x B6.H2g7)F1, but not other candidates, developed T1D. Furthermore, CD4 T cells from these mice strongly promoted diabetogenic AI4 T cell activation when co-transferred into NOD.Rag1-/- mice. In contrast, CD4 T cells from St6galnac1-intact (B6.H2g7.Idd32NOD/NOD x B6.H2g7)F1 mice suppressed AI4 activation and T1D development. In summary, we have identified a novel, recessively-acting T1D susceptibility gene hidden in the normally T1D-resistant B6 mouse strain, with striking effects on pathogenic CD8 T cell activation. Our results may aid the screening of humans at future risk for T1D as well as provide a target for possible disease interventions.

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Determining how much ATP is yielded from fermentation by rumen microbes

Bo Zhang, Courtney McCourt, and Timothy J. Hackmann

Department of Animal Sciences, University of Florida, Gainesville, FL

Rumen microbes represent the largest and cheapest source of protein for cattle, making it a perennial goal of animal nutrition to increase microbial protein production. However, both total yield and efficiency of microbial yield are low, and it is uncertain how inefficient microbes are. We must know the ATP yield of fermentation in order to determine the microbial growth efficiency accurately. The ATP yield from fermentation is thought to be well known, but recent genomic evidence suggests fermentation pathways yield more ATP than expected. Some bacteria encode two ion pumps (Ech, Rnf) that, in conjunction with ATP synthase, raise ATP yield by 50% in the pathway of fermentation of glucose to butyrate. Some bacteria that encode Rnf pump raise ATP yield by 12.5% in the pathway that forming acetate and propionate. Additionally, the existence of an unconventional glycolytic enzyme Pfp raises ATP yield by sparing ATP because this enzyme uses pyrophosphate rather than ATP when transferring fructose-6P to fructose-1,6P2. Here, we will determine if this genomic evidence is supported experimentally for bacteria and protozoa fermenting glucose. Specifically, we will show our current finding about a new pathway for acetate formation in bacteria, and discuss how to verify and determine the activities of Ech, Rnf, and Pfp in bacteria and protozoa isolated from the rumen. By determining the activity of these enzymes (Ech, Rnf, Pfp) in rumen bacteria and protozoa, we can determine the accurate ATP yield from major fermentation pathway of glucose and reveal how inefficient microbes are.

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Effects of dietary vitamin D3 or 25-hydroxyvitamin D3 on mineral metabolism in growing calves R. Zimpel1, M. B. Poindexter1, A. Vieira-Neto1, A. Husnain1, S. Buoniconti1, C.D. Nelson1, J. E. P. Santos1

1University of Florida, Gainesville, FL, 32611 Objectives were to determine the effects of dose and source of vitamin D (vitamin D3 or 25-hydroxyvitamin D3 [25OHD3]) on mineral metabolism and bone mineral density (BMD) in calves. The hypothesis was that dietary 25OHD3 would enhance Ca accretion and BMD. Forty-five Holstein male calves were enrolled within the first week of age in a randomized complete block design. Calves were assigned to 1 of 5 treatments: T1 (0.25 g of vitamin D3/kg BW), T2 (0.25 g of vitamin D3 + 1.5 g of 25OHD3/kg BW), T3 (0.25 g of vitamin D3 + 3.0 g of 25OHD3/kg BW), T4 (1.75 g of vitamin D3/kg BW) and T5 (3.25 g of vitamin D3/kg BW). Treatments were supplied from birth to 131 ± 9 d of age when calves were euthanized, and tissues collected. Calves were fed milk replacer until 49 d of age and had ad libitum access to starter grain and water. Blood was sampled on d -15, -14, -13, -12, -11, -9, -7, -5, -3 and -1 relative to slaughter. Intake of DM was evaluated in the last 15 d before slaughter. Total fecal collection and spot urine was sampled on d -4 to -2 relative to slaughter. The BMD was determined in the right metacarpus. Data were analyzed by ANOVA with mixed models using the MIXED procedure of SAS. Contrasts evaluated included the effects of supplementing vitamin D (Sup: T1 vs. T2+T3+T4+T5), source of vitamin D (Source: T2+T3 vs. T4+T5), dose of vitamin D (Dose: T2+T4 vs. T3+T5), or the interaction between Source and Dose (Int: T2 + T5 vs. T3 + T4). The weight of liver, pancreas and thyroid gland, and weight and dimensions of kidneys and spleen did not differ among treatments. Intake of DM or Ca did not differ among treatments.  Supplementing vitamin D reduced blood Ca; however, supplemental 25OHD3 increased blood Ca compared with vitamin D3. Treatment did not affect digestibility or retention of Ca, but calves fed 25OHD3 tended to have increased BMD. Item T1 T2 T3 T4 T5 SE Body weight, kg 146.4 148.5 143.3 145.9 150.1 8.7 BW gain, kg/d Intake

1.32 1.20 1.19 1.25 1.34 0.12

DM, kg/d 4.3 4.0 4.0 4.0 4.3 0.25 Ca, g/d 61.9 55.1 58.6 56.7 62.2 4.0

Blood tCa, mM *,† 2.868 2.858 2.840 2.788 2.808 0.024 iCa, mM * 1.473 1.454 1.437 1.446 1.429 0.014 Calcitriol, pg/mL 51.4 53.8 55.2 53.4 57.0 3.2

BMD, g/cm2 †,‡ 0.986 1.033 1.054 0.967 1.038 0.024 Ca digestibility, % 50.5 53.2 51.6 52.1 52.9 2.5 Ca retention, g/d 31.1 29.0 30.3 29.4 33.1 2.9 * Sup (P<0.09); † Source (P<0.10); ‡ Dose (P<0.07). Bone density, calcium, vitamin D

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